No / Mechanochemical chemoenzymatic peptide and amide bond formation catalysed by papain was studied by ball milling. Despite the high-energy mixing experienced inside the ball mill, the biocatalyst proved stable and highly efficient to catalyse the formation of α,α- and α,β-dipeptides. This strategy was further extended to the enzymatic acylation of amines by milling, and to the mechanosynthesis of a derivative of the valuable dipeptide L-alanyl-L-glutamine. / We thank RWTH Aachen University for support from the Distinguished Professorship Program funded by the Excellence Initiative of the German federal and state governments. EPSRC, grant no. EP/L019655/1.

INTRODUÇÃO: O modelo eslastase-induzido de aneurismas tem se destacado, nos últimos anos, porque simula as características geométricas dos aneurismas intracranianos humanos. A elastase destrói as fibras elásticas e dilata as artérias. A papaína é uma enzima que ainda não foi usada com esta finalidade. Os objetivos deste estudo foram determinar se a papaína produz aneurismas saculares em coelhos, e comparar suas características macroscópicas e histológicas com as dos aneurismas elastase-induzidos. MÉTODO: Dezoito coelhos brancos Nova Zelândia (1,9-3,2 kg) foram divididos em 3 grupos: I- elastase (n=7), II- papaína (n=8) e III- cirurgia controle (n=3). Os animais foram submetidos à exposição cirúrgica do pescoço, sendo que a artéria carótida comum direita foi usada como teste e a artéria carótida comum esquerda como controle. No 21° dia após a cirurgia, os animais foram sacrificados para retirada das artérias, tomada de suas medidas e análise histológica. Considerou-se formação de aneurisma quando a artéria teste dilatou em relação ao seu controle. RESULTADOS: Não houve aneurismas no grupo cirúrgico controle. Houve formação de aneurismas nos grupos elastase (71,4%) e papaína (100%). A diferença do diâmetro das artérias testes e seus respectivos controles não foi significativa (p= 0,15) entre os grupos elastase (média= 1,2 ± 0,4mm) e papaína (média= 2,1 ± 0,4mm), embora houvesse tendência deste último à maior dilatação . A histologia demonstrou que a papaína produziu maior tendência à lesão endotelial, à trombose (p = 0,01) e à inflamação parietal do que a elastase. A análise da fibrose intimal foi prejudicada em 50% dos casos do grupo papaína devido à trombose acentuada. Não houve diferença significativa entre os grupos quanto ao espessamento parietal (p=0,81) e ao grau de destruição das fibras elásticas (p= 0,009). CONCLUSÕES: A papaína produz aneurismas com tamanhos semelhantes aos da elastase, contudo a papaína provoca maior lesão endotelial, maior trombose e maior inflamação do que a elastase / INTRODUCTION: The elastase-induced model of experimental saccular aneurysms has been relevant in the last years because it mimics the size and geometric features of human intracranial aneurysms. Elastase destroys the arterys elastic fibers and produces arterial enlargement. Papain enzyme is also elastolytic but it had not been tested on saccular aneurysms creation yet. The purpose of this study was determine if papain produces saccular aneurysms in rabbits and to compare its gross and microscopic features are with the elastaseinduced aneurysms. METHODS: Eighteen New Zealand white rabbits (1.9 kg 3,2 kg) were separated in 3 groups: 1) sham (n=3) 0,9% saline solution; papain (n=8) 17-40 U; elastase (n=7) 6-8 U. The animals underwent surgical exposure of the neck; the right common carotid artery (RCCA) was used as the test and the left common carotid artery (LCCA) as the control. On the 21st day after surgery, animals were sacrificed for removal of the arteries, measurements and histological analysis. We determine formation of aneurysm to occur when the test artery dilated compared to the control. RESULTS: The sham group didnt develop aneurysms. There was aneurysm formation in the elastase (71,4%) and papain (100%) groups. The difference of the diameter of the tests and their respective controls is not significant (p=0,15) between elastase (average = 1,2 ± 0,4 mm) and papain (average = 2,1 ± 0,4mm) groups although there was tendency of this last one to produce larger aneurysms. the and to thrombosis (p = 0,01) and to parietal inflammation than the elastase. The analysis of the intimal fibrosis was not possible in 50% of papain cases due to pronounced thrombosis. There was no significant difference between the groups regarding the parietal thickening (p = 0,81) and the degree of destruction of the elastic fibers (0,009). CONCLUSION: Papain creates saccular aneurysms with similar dimensions to elastase-induced aneurysms. The microscopic results indicated papain destroys more endothelial cells, produces more thrombosis and more inflammatory process than elastase

A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Papain is a proteolytic enzyme applied for wound healing and debridement. However, stability issues as well as allergenic reactions reported by patients submitted to papain pharmaceutics led to restriction of papain containing products for topical use by international regulatory agencies. This work aimed the development of nanostructured hydrogel containing papain and cyclodextrin complex or papain in order to obtain stable and suitable pharmaceutical form as a wound dressing with reduced allergenic properties. The hydrogel synthesis was performed by combining freezing cycles and the simultaneous crosslinking and sterilization process promoted by gamma irradiation to achieve a nanostructured hydrogel for the loading of the papain and cyclodextrin complex. The biological and physical-chemical properties of the complex and the final product were assayed. The PVA based hydrogel containing cyclodextrin-papain complex presented desirable characteristics for wound dressing purposes. In addition, an in vitro shift in the immunological response and an increase in the cytocompatibility if compared to native papain were observed as a function of the molecular encapsulation with cyclodextrin. The process of irradiation was not capable of altering papain cytotoxicity, but conferred a slight decrease in the immunogenic properties. In conclusion, the developed hydrogel was promising as a novel papain containing dressing with potential reduced adverse reactions.

A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Papain is a proteolytic enzyme applied for wound healing and debridement. However, stability issues as well as allergenic reactions reported by patients submitted to papain pharmaceutics led to restriction of papain containing products for topical use by international regulatory agencies. This work aimed the development of nanostructured hydrogel containing papain and cyclodextrin complex or papain in order to obtain stable and suitable pharmaceutical form as a wound dressing with reduced allergenic properties. The hydrogel synthesis was performed by combining freezing cycles and the simultaneous crosslinking and sterilization process promoted by gamma irradiation to achieve a nanostructured hydrogel for the loading of the papain and cyclodextrin complex. The biological and physical-chemical properties of the complex and the final product were assayed. The PVA based hydrogel containing cyclodextrin-papain complex presented desirable characteristics for wound dressing purposes. In addition, an in vitro shift in the immunological response and an increase in the cytocompatibility if compared to native papain were observed as a function of the molecular encapsulation with cyclodextrin. The process of irradiation was not capable of altering papain cytotoxicity, but conferred a slight decrease in the immunogenic properties. In conclusion, the developed hydrogel was promising as a novel papain containing dressing with potential reduced adverse reactions.

As úlceras por pressão são definidas como lesões causadas pela constante pressão exercida sobre um determinado ponto do corpo, ocasionando comprometimento do aporte sangüíneo com diminuição ou interrupção da irrigação tissular, ocasionando oclusão de vasos e capilares, isquemia e morte celular. O objetivo principal deste trabalho foi verificar a resposta da cicatrização em úlceras por pressão, em clientes no atendimento domiciliar com a estimulação do ultra-som pulsado de baixa intensidade. A metodologia proposta foi a investigação de voluntários humanos não diabéticos, portadores de úlceras por pressão de qualquer grau, região, tamanho e profundidade em qualquer fase de cicatrização, sem processo infeccioso evidente, sem uso de medicação antiinflamatória. Todas as lesões receberam assistência de enfermagem obedecendo ao mesmo protocolo de curativos, para os portadores de 2 lesões apenas uma delas recebeu estimulação de ultra-som pulsado de baixa intensidade 30 mW/\'CM POT.2\', freqüência 1.5 Mhz em sessões de 20 minutos, três vezes por semana, durante seis meses, perfazendo um ciclo de aproximadamente 72 aplicações, ou menos sessões quando a alta aconteceu antes do prazo. Os resultados foram acompanhados através de quatro instrumentos: análise comparativa descritiva, análise histológica, análise morfométrica e análise fotográfica . A pesquisa foi observacional descritiva, sendo realizada uma análise estatística, através de dados extraídos dos prontuários e mensurações das áreas das lesões. O estudo histológico revelou melhora na vascularização, demonstrou angiogênese e processo inflamatório local. / The pressure ulcers are areas of injured skin and tissue caused by constant pressure on certain areas of the body, compromising the irrigation of tissue and also, causing the occlusion of vases and capillaries, ischemia and cellular death. The main objective of this work was the evaluation the wound healing process in pressure ulcers, during homecare service, with stimulation of low intensity ultrasound combined with papain. The methodology evaluation was applied in human volunteers without diabetic, with wounds in any ranges, region, size and depth. These wounds were in any phase and without any evident infectious process, without use of any antiinflammatory drugs. During all the execution of the work the injuries were treated according to the same protocol . Patients with only one wound received ultrasound stimulation in sessions of twenty minutes, three times per week, during six months, constituting a healing cycle of approximately 72 applications. Early treatment closing caused a reduction of sessions number. Considering patients with two injuries only one of the received the ultrasonic stimulation. The results had been followed through four studies: descriptive comparative analysis, histological analysis, morphometric analysis and photographic analysis. The results obtained were submitted to statistical analysis, by analyzing data extracted from clinical records and measurements of injury areas. The histological study disclosed improvement in the vascularization, it demonstrated angiogenesis and local inflammatory process.

As úlceras por pressão são definidas como lesões causadas pela constante pressão exercida sobre um determinado ponto do corpo, ocasionando comprometimento do aporte sangüíneo com diminuição ou interrupção da irrigação tissular, ocasionando oclusão de vasos e capilares, isquemia e morte celular. O objetivo principal deste trabalho foi verificar a resposta da cicatrização em úlceras por pressão, em clientes no atendimento domiciliar com a estimulação do ultra-som pulsado de baixa intensidade. A metodologia proposta foi a investigação de voluntários humanos não diabéticos, portadores de úlceras por pressão de qualquer grau, região, tamanho e profundidade em qualquer fase de cicatrização, sem processo infeccioso evidente, sem uso de medicação antiinflamatória. Todas as lesões receberam assistência de enfermagem obedecendo ao mesmo protocolo de curativos, para os portadores de 2 lesões apenas uma delas recebeu estimulação de ultra-som pulsado de baixa intensidade 30 mW/\'CM POT.2\', freqüência 1.5 Mhz em sessões de 20 minutos, três vezes por semana, durante seis meses, perfazendo um ciclo de aproximadamente 72 aplicações, ou menos sessões quando a alta aconteceu antes do prazo. Os resultados foram acompanhados através de quatro instrumentos: análise comparativa descritiva, análise histológica, análise morfométrica e análise fotográfica . A pesquisa foi observacional descritiva, sendo realizada uma análise estatística, através de dados extraídos dos prontuários e mensurações das áreas das lesões. O estudo histológico revelou melhora na vascularização, demonstrou angiogênese e processo inflamatório local. / The pressure ulcers are areas of injured skin and tissue caused by constant pressure on certain areas of the body, compromising the irrigation of tissue and also, causing the occlusion of vases and capillaries, ischemia and cellular death. The main objective of this work was the evaluation the wound healing process in pressure ulcers, during homecare service, with stimulation of low intensity ultrasound combined with papain. The methodology evaluation was applied in human volunteers without diabetic, with wounds in any ranges, region, size and depth. These wounds were in any phase and without any evident infectious process, without use of any antiinflammatory drugs. During all the execution of the work the injuries were treated according to the same protocol . Patients with only one wound received ultrasound stimulation in sessions of twenty minutes, three times per week, during six months, constituting a healing cycle of approximately 72 applications. Early treatment closing caused a reduction of sessions number. Considering patients with two injuries only one of the received the ultrasonic stimulation. The results had been followed through four studies: descriptive comparative analysis, histological analysis, morphometric analysis and photographic analysis. The results obtained were submitted to statistical analysis, by analyzing data extracted from clinical records and measurements of injury areas. The histological study disclosed improvement in the vascularization, it demonstrated angiogenesis and local inflammatory process.

Chemotherapy is the first line of treatment used in cancer. Chemotherapy drugs such as cisplatin, carboplatin and oxaliplatin are used in treatment. Cisplatin enters the cell through copper transporter CTR1 by passive diffusion and bind to DNA and proteins. Cisplatin is found to inhibit several enzymes targeting cysteine, histidine and methionine residues, which are expected to be responsible for its anticancer activity. A better understanding of how the size and shape and leaving ligands of platinum complexes affect cysteine protease, papain enzyme are studied. This could give new ways to optimize anticancer activity. The activity of papain enzyme was measured on UV-Visible spectroscopy. The inhibition profile of papain with different platinum (II) complexes, and with different combinations was studied.

Beef fajitas have become a popular food item, but the supply of traditional muscles is insufficient to meet the growing demand. There is a need for alternative muscles that have similar eating characteristics to those currently marketed as beef fajitas. Four different treatments - papain (P), blade tenderization (B), papain + blade tenderization (P+B), and control (C) - were applied to sixty USDA Choice M. diaphragma pars costalis, M. transversus abdominis, M. obliquus abdominis internus, M. rhomboideus, M. trapezius, M. latissimus, and M. serratus ventralis. Muscles were cut into sections and frozen at -10ºC for evaluation by a trained sensory panel, consumer panel, and Warner-Bratzler shear force (WBSF) measurements. Trained panelists found that regardless of muscle, the addition of papain improved palatability scores. In general, treatment tended not to affect the palatability scores of the M. diaphragma pars costalis and M. serratus ventralis, which tended to receive higher scores in comparison to the other muscles. In general, the control and blade tenderized M. trapezius received the lowest trained and consumer panel palatability scores and had the highest WBSF values (P < 0.05). Regardless of muscle (except for M. diaphragma pars costalis and M. serratus ventralis), P and P+B treatments reduced WBSF values (P < 0.05). Consumers (n=81) gave the M. transversus abdominis, M. serratus ventralis, and M. latissimus similar (P > 0.05) tenderness ratings when the P treatment was applied. Consumers tended to prefer the flavor and tenderness of beef fajita strips that were treated with P and P+B and indicated a preference to purchase muscles with these treatments. Consumers were willing to purchase M. serratus ventralis treated with P+B and M. latissimus treated with P the majority of the time. Papain improved the eating quality of the muscles studied. The M. latissimus and M. serratus ventralis when treated with papain alone or in combination with blade tenderization, performed well enough to be considered as alternatives for traditional beef fajitas.

Orientador: Celso Koogi Sonoda / Banca: Sônia Regina Panzarini Barioni / Banca: Wilson Roberto Poi / Banca: Fábio Yoshio Tanaka / Banca: André Dotto Sottovia / Resumo: Para um bom prognóstico no reimplante dentário, é necessário que o dente seja reposicionado o mais rápido possível em seu alvéolo de origem ou mantido em meio de conservação adequado. Quando o dente avulsionado é mantido em condições inadequadas de conservação, a remoção do ligamento periodontal desvitalizado é a conduta mais apropriada para minimizar a ocorrência da reabsorção radicular. Com o objetivo de prevenir e/ou retardar a instalação de reabsorções radiculares e preservar a integridade da camada de cemento propõe-se o estudo do processo de reparo em dentes de ratos reimplantados tardiamente, após a utilização da solução de papaína e fluoreto de sódio no tratamento de superfície radicular. Essa proposta tem como base as propriedades da papaína de remoção do tecido necrótico, ação antioxidante e antimicrobiana. Foram utilizados os incisivos superiores direitos de 40 ratos, divididos em 4 grupos, sendo que no Grupo I os dentes foram imediatamente reimplantados em seus respectivos alvéolos. Nos grupos II, III, IV, os dentes extraídos foram mantidos em meio seco pelo período de 1 hora. No grupo II, os dentes foram imersos em solução de papaína a 50% por 20 minutos, friccionados com gaze por 1 minuto e imerso em fluoreto de sódio fosfato acidulado a 2%, pH 5, pelo período de 20 minutos. No grupo III, os dentes foram mantidos em soro fisiológico por 20 minutos, tiveram a superfície radicular friccionada por 1 minuto e imerso em fluoreto de sódio fosfato acidulado a 2%, pH 5, por um período de 20 minutos. No grupo IV, os dentes foram reimplantados em seus respectivos alvéolos, sem nenhum tratamento a superfície radicular. Previamente ao reimplante, a polpa radicular foi removida e os canais preenchidos com Ca(OH)2 veiculado em propilenoglicol. Após o reimplante, os animais receberam antibioticoterapia sistêmica... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: For a good prognostic in tooth replanting, it is necessary that the tooth be repositioned in its socket as quickly as possible or otherwise be properly conserved. When the avulsed tooth is improperly conserved, removing the devitalized periodontal ligament is the appropriate conduct to minimize the occurrence of radicular reabsorption. The aim of this study was to search for an effective form of the above process, while preserving the integrity of the cement layer. In this case, papain was used, due to its necrotic tissue removing proprieties and its microbacterial action. The research was conducted with 40 extracted rat incisives, divided into 4 groups. In group I the teeth were replanted immediately in their respective sockets. In groups II, III and IV the extracted teeth were kept in a dry environment for a period of 1 hour. In group II, the teeth were immersed in papain solution (50%) for 20 minutes, rubbed with gauze for 1 minute and immersed in sodium fluoride (2%, pH 5) for a period of 20 minutes. In group III, the teeth were kept in a saline solution for 20 minutes, the radicular surface was rubbed for 1 minute, and they were immersed in sodium fluoride (2%, pH5) for 20 minutes. In group IV, the teeth were replanted in their respective sockets without any treatment. Before replanting, the radicular pulp was removed and the root filled with Ca(OH)2 dissolved in propilenoglicol. Immediately upon replanting the rats received antibiotic systemic therapy and after 60 days they were euthanized. The pieces containing the replanted incisive were subjected to histological analysis. The results show a higher occurrence of ankylosis in group II when compared to group I (p<0.05). Reabsorption by substitution occurs more in group IV when compared to group I (p<0.05) and II (p<0.001). Considering all areas of reabsorption, it was observed that the teeth in group... (Complete abstract click electronic access below) / Doutor

Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.